Method of producing solution containing hemoglobin a2, method of producing solution containing hemoglobin a2 and hemoglobin f, and method of producing hemoglobin quality control material

Abstract

To provide a method of producing a solution containing hemoglobin A2, which enables preparation of a hemoglobin A2-rich solution from an easily available blood sample, a method of producing a solution containing hemoglobin A2 and hemoglobin F, and a method of producing a hemoglobin quality control material.SOLUTION: Provided herein are: a method of producing a solution containing hemoglobin A2, the method comprising bringing a hemoglobin-containing solution with a pH of 7.8 to 9.5 into contact with an anion exchanger; and a method of producing a hemoglobin quality control material.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for producing a solution containing hemoglobin A2, a method for producing a solution containing hemoglobin A2 and hemoglobin F, and a method for producing a quality control substance for hemoglobin. [Background technology]

[0002] In the manufacture of quality control materials used as standards in the quantification of hemoglobin (hereinafter also referred to as "Hb"), human blood is generally used as a raw material. The percentages of Hb contained in normal adult blood are HbA at about 95-96%, HbA2 at about 2-3%, and HbF at about 0.5-1.5%. On the other hand, since it is desirable to prepare quality control materials having Hb concentrations above the clinical judgment value in addition to samples having Hb concentrations below the clinical judgment value, the Hb concentration in the quality control materials may be set higher than the Hb concentration in normal adult blood. For example, HbF is present in umbilical cord blood at a ratio of about 85% of the total Hb, and therefore umbilical cord blood is generally used to prepare HbF control samples. Patent Document 1 describes a control sample of HbF containing a hemolysate of human red blood cells, a mixed hemolysate of human umbilical cord blood, and sucrose. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2016-075504 A Summary of the Invention [Problem to be solved by the invention]

[0004] Because the blood of β-thalassemia patients contains a high proportion of HbA2, the blood of β-thalassemia patients is sometimes used as a raw material in the production of HbA2 control samples. However, since it is difficult to obtain blood from β-thalassemia patients, the challenge in the production of HbA2 control samples is how to obtain a solution with a high proportion of HbA2.

[0005] In view of this situation, the present disclosure relates to a method for producing a solution containing HbA2, which enables a solution with a high proportion of HbA2 to be obtained from easily available blood samples, a method for producing a solution containing HbA2 and HbF, and a method for producing a quality control substance for Hb. [Means for solving the problem]

[0006] Means for solving the above problems include the following aspects. <1> A method for producing a solution containing hemoglobin A2, comprising contacting a solution containing hemoglobin and having a pH of 7.8 to 9.5 with an anion exchanger. <2> and recovering the solution after contacting the anion exchanger. <1> A method for producing a solution containing hemoglobin A2 according to claim 1. <3> The pH of the solution containing hemoglobin is 8.0 to 9.0. <1> or <2> A method for producing a solution containing hemoglobin A2 according to claim 1. <4> The ratio of hemoglobin A2 to the total amount of hemoglobin is increased in the solution after contact with the anion exchanger, compared to the solution before contact with the anion exchanger. <1> ~ <3> 2. A method for producing a solution containing hemoglobin A2 according to any one of claims 1 to 11. <5> The hemoglobin-containing solution having a pH of 7.8 to 9.5 contains hemoglobin derived from the blood of a human subject without hemoglobin abnormalities and a buffer solution. <1> ~ <4> 2. A method for producing a solution containing hemoglobin A2 according to any one of claims 1 to 11. <6> <1> ~ <5> 2. A method for producing a solution containing hemoglobin A2 and hemoglobin F, comprising mixing a solution containing hemoglobin A2 obtained by any one of the production methods described above with a solution containing hemoglobin F. <7> A method for producing a quality control substance for hemoglobin, comprising contacting a solution containing hemoglobin and having a pH of 7.8 to 9.5 with an anion exchanger. <8> and recovering the solution after contacting the anion exchanger. <7> A method for producing a quality control material for hemoglobin according to claim 1. <9> The pH of the solution containing hemoglobin is 8.0 to 9.0. <7> or <8> A method for producing a quality control material for hemoglobin according to claim 1. <10> The ratio of hemoglobin A2 to the total amount of hemoglobin is increased in the solution after contact with the anion exchanger, compared to the solution before contact with the anion exchanger. <7> ~ <9> 13. A method for producing a quality control substance for hemoglobin according to any one of claims 1 to 12. <11> The hemoglobin-containing solution having a pH of 7.8 to 9.5 contains hemoglobin derived from the blood of a human subject without hemoglobin abnormalities and a buffer solution. <7> ~ <10> 13. A method for producing a quality control substance for hemoglobin according to any one of claims 1 to 12. <12> The method further comprises mixing the solution obtained by contacting the anion exchanger with a solution containing hemoglobin F. <7> ~ <11> 13. A method for producing a quality control substance for hemoglobin according to any one of claims 1 to 12. Effect of the Invention

[0007] According to the present disclosure, there are provided a method for producing a solution containing HbA2, a method for producing a solution containing HbA2 and HbF, and a method for producing a quality control substance for Hb, which are capable of obtaining a solution with a high proportion of HbA2 from easily available blood samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Hereinafter, the form for carrying out the embodiment of the present disclosure will be described in detail. However, the embodiment of the present disclosure is not limited to the following embodiment. In the following embodiment, the components (including element steps, etc.) are not essential unless specifically stated. The same applies to the numerical values ​​and their ranges, and they do not limit the embodiment of the present disclosure.

[0009] In the present disclosure, the term "step" includes not only a step that is independent of other steps, but also a step that cannot be clearly distinguished from other steps as long as the purpose of the step is achieved.

[0010] In the present disclosure, a numerical range indicated using "~" includes the numerical values ​​before and after "~" as the minimum and maximum values, respectively.

[0011] In the numerical ranges described in the present disclosure in stages, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. In addition, in the numerical ranges described in the present disclosure, the upper or lower limit value of the numerical range may be replaced with a value shown in the examples.

[0012] In the present disclosure, the total amount of Hb in a solution is measured by the cyanmethemoglobin method, specifically, for example, using Hemoglobin Test Wako (FUJIFILM Wako Pure Chemical Industries, Ltd.).

[0013] In the present disclosure, the proportions of HbA2 and HbF in Hb are measured by the peak area ratio in high performance liquid chromatography. High performance liquid chromatography is performed under the following conditions or equivalent conditions. The equivalent conditions refer to conditions that do not substantially affect the elution power of Hb and each Hb species (for example, the error in measuring the proportions of HbA2 and HbF is ±0.5% or less). The apparatus used is an ADAMS HA-8180T (Arkray, Inc.). The apparatus uses a column unit 80T (Arkray, Inc.), which is a column packed with 0.45 ml of a hydrophilic polymer made of a methacrylic acid ester copolymer as a packing material. The reagents used are 80A as eluent A for eluting Hb from the column, 80B as eluent B, and 80CT (Arkray, Inc.) as eluent C, and 80H as a hemolysis / washing solution. The compositions and pH of 80A, 80B, 80CT, and 80H are as follows. These reagents are passed through the column at a flow rate of 1.7 ml / min. Eluent A (80A): Phosphate buffer (pH 5.35 ± 0.05) Eluent B (80B): Phosphate buffer (pH 8.05 ± 0.20) Eluent C: (80CT): Phosphate buffer (pH 7.0-7.4) Hemolysis and washing solution (80H): Phosphate buffer (pH 7.5 ± 0.1) containing surfactant

[0014] <Method for producing a solution containing HbA2> The method for producing a solution containing HbA2 according to an embodiment of the present disclosure includes contacting a solution of pH 7.8 to 9.5 containing Hb with an anion exchanger (hereinafter, also referred to as the "contact step"). Hereinafter, the solution containing Hb before contacting with the anion exchanger is also referred to as the "Hb solution", and the solution containing HbA2 obtained after contacting with the anion exchanger is also referred to as the "HbA2 solution". Note that the obtained HbA2 solution may contain components other than HbA2 (for example, other Hbs such as HbA0, HbA1, HbF, buffer, stabilizer, etc.).

[0015] The inventors found that when a Hb solution obtained from normal adult blood is purified with an anion exchanger, the proportion of HbA2 in total Hb is high when the solution is at a specific pH. This phenomenon is described in detail below. Since the isoelectric point of Hb is about 7, when the pH of the Hb solution is high, Hb is anionized and the binding ability between Hb and the anion exchanger is enhanced. That is, as the pH of the Hb solution increases, the amount of Hb remaining in the solution without binding to the anion exchanger decreases. Also, it is considered that the amount of HbA2 remaining in the solution decreases as the pH increases. However, when the Hb solution is at a specific pH, it has been found that as the pH increases, the proportion of HbA2 in total Hb remaining in the solution without binding to the anion exchanger increases. This is presumably because when the Hb solution is in a specific pH range, Hb other than HbA2 preferentially binds to the anion exchanger, resulting in a relative increase in the HbA2 concentration. However, the embodiments of the present disclosure are not limited to the above presumed mechanism at all.

[0016] According to the method for producing an HbA2 solution of the present disclosure, for example, an HbA2 solution having a high HbA2 ratio can be prepared from the blood of a normal adult. Therefore, there is no need to use blood having a high HbA2 ratio (e.g., blood of a β-thalassemia patient). According to the method for producing an HbA2 solution of the present disclosure, an HbA2 solution may be prepared from blood having a high HbA2 ratio (e.g., blood of a β-thalassemia patient), or from blood having a normal or lower than normal HbA2 ratio.

[0017] Typically, the ratio of HbA2 to the total amount of Hb in an Hb solution after contact with an anion exchanger is increased compared to the Hb solution before contact with the anion exchanger.

[0018] [Contact process] In the contact step, the Hb solution at pH 7.8 to 9.5 is brought into contact with the anion exchanger. The origin of the Hb solution is not particularly limited, and examples thereof include human and non-human animal blood, with human blood being preferred. Examples of non-human animals include non-human mammals (monkeys, dogs, cats, mice, rats, rabbits, cows, horses, pigs, sheep, etc.) and birds (chickens, quails, etc.). It is preferred to use an Hb solution obtained by extracting Hb from the blood of the human or non-human animal and purifying it as necessary. Peripheral blood is preferred as the blood, from the viewpoint of easy acquisition.

[0019] For example, the Hb solution may be prepared by hemolyzing the blood of the human or non-human animal and extracting Hb. Hemolysis may be performed, for example, by adding a buffer solution to the precipitate obtained by centrifuging whole blood obtained by blood collection, and freezing and thawing the precipitate. Alternatively, the solution obtained by hemolysis as described above may be centrifuged to recover the supernatant to prepare the Hb solution. Furthermore, the supernatant recovered as described above may be purified by dialysis or the like to prepare the Hb solution.

[0020] The concentration of Hb in the Hb solution when it is contacted with the anion exchanger is preferably 28 to 41 g / L, more preferably 32 to 37 g / L, and even more preferably 34 to 35 g / L.

[0021] The ratio of HbA2 to the total Hb in the Hb solution when contacted with the anion exchanger is preferably 1.0% or more, more preferably 1.5% or more, and even more preferably 2.0% or more, from the viewpoint of efficient concentration of HbA2. The ratio may be 5.0% or less, 4.0% or less, or 3.0% or less. Therefore, the ratio may be 1.0 to 5.0%, 1.5 to 4.0%, or 2.0 to 3.0%.

[0022] The Hb solution preferably contains a buffer solution to adjust the pH, such as Tris buffer solution (Tris-HCl, etc.), carbonate buffer solution, HEPES buffer solution, phosphate buffer solution, etc.

[0023] The pH of the Hb solution when contacted with the anion exchanger is 7.8 to 9.5. From the viewpoint of suppressing the binding of HbA2 to the anion exchanger and improving the recovery amount of HbA2, the pH of the Hb solution is preferably 9.0 or less. From the viewpoint of increasing the ratio of HbA2 in the obtained HbA2 solution, the pH of the Hb solution is preferably 8.0 or more, more preferably 8.3 or more, even more preferably 8.5 or more, and particularly preferably 8.8 or more. From the above viewpoints, the pH of the Hb solution is preferably 8.0 to 9.0, more preferably 8.3 to 9.0, even more preferably 8.5 to 9.0, and particularly preferably 8.8 to 9.0. In the present disclosure, pH refers to the pH at 25°C.

[0024] In one embodiment, the Hb solution includes Hb derived from the blood of a human subject without Hb abnormality and a buffer solution. Here, "Hb abnormality" includes abnormality due to a higher or lower amount of Hb compared to the blood of a normal human subject, abnormality due to a higher or lower ratio of HbA2 in total Hb compared to the blood of a normal human subject, and other structural and / or functional abnormalities of Hb, and preferably refers to abnormality due to a higher or lower ratio of HbA2 in total Hb compared to the blood of a normal human subject. An abnormality in the amount of Hb compared to the blood of a normal human subject can be determined by whether or not the Hb concentration in the blood deviates from a predetermined normal range. Similarly, an abnormality in the ratio of HbA2 in total Hb compared to the blood of a normal human subject can be determined by whether or not the ratio of HbA2 deviates from a predetermined normal range. Examples of the buffer solution include the above-mentioned buffer solutions.

[0025] Examples of anion exchangers include strongly basic anion exchange resins and weakly basic anion exchange resins. Strongly basic anion exchange resins include anion exchange resins having quaternary amine exchange groups (trimethylammonium groups, dimethylethanolammonium groups, etc.) as ion exchange groups. Weakly basic anion exchange resins include anion exchange resins having tertiary amine exchange groups (polyamines, dimethylamines, etc.), secondary amine exchange groups, primary amine exchange groups, etc. as ion exchange groups. From the viewpoint of being able to exchange ions in a wide pH range, strongly basic anion exchange resins are preferred, and anion exchange resins having quaternary amine exchange groups are more preferred. Commercially available anion exchange resins may be used. Examples of commercially available anion exchange resins include Macro-PrepHighQ (Biorad).

[0026] The manner of contacting the Hb solution with the anion exchanger is not particularly limited. For example, the Hb solution may be mixed with the anion exchanger, or the Hb solution may be added to a column packed with the anion exchanger, the former being convenient.

[0027] [Other steps] The method for producing an HbA2 solution of the present disclosure may include other steps in addition to the contacting step. For example, the method for producing an HbA2 solution may include recovering the solution after contacting with an anion exchanger. That is, the method for producing an HbA2 solution may include recovering the HbA2 solution after contacting with an anion exchanger by separating it from the anion exchanger. As described above, when a solution containing Hb and having a pH of 7.8 to 9.5 is contacted with an anion exchanger, the ratio of HbA2 to the total amount of Hb in the solution increases relatively, so that by recovering the solution, a solution with a high HbA2 concentration can be selectively recovered.

[0028] A simple method for collecting the Hb is to precipitate the anion exchanger by centrifugation and collect the supernatant. Alternatively, if the Hb solution is added to the anion exchanger packed in a column, the flow-through may be collected.

[0029] The method for producing the HbA2 solution may further include contacting the Hb solution with an anion exchanger to recover the HbA2 solution, washing the anion exchanger with a buffer solution, and then recovering the supernatant (or flow-through). The washing operation allows recovery of HbA2 that was not recovered in one HbA2 solution recovery, and the recovery amount can be increased. The number of washes may be one or two or more, for example, 1 to 10 times, 1 to 5 times, or 1 to 3 times.

[0030] The method for producing an HbA2 solution may further include contacting the recovered HbA2 solution with an anion exchanger again and then recovering the solution, i.e., contacting the solution with the anion exchanger may be repeated.

[0031] The method for producing the HbA2 solution may include a step of concentrating the HbA2 solution obtained by contacting the Hb solution with an anion exchanger. The concentration may be performed, for example, by precipitating proteins in the HbA2 solution with ammonium sulfate, collecting the precipitate by centrifugation, and adding a buffer solution.

[0032] The method for producing the HbA2 solution may include a step of further purifying the HbA2 solution obtained by contacting the Hb solution with an anion exchanger. The step of further purifying may be performed after the step of concentrating the HbA2 solution. Purification can be performed by dialysis or the like.

[0033] <Method for Producing a Solution Containing HbA2 and HbF> The method for producing a solution containing HbA2 and HbF in one embodiment of the present disclosure includes mixing the HbA2 solution obtained by the aforementioned method for producing the HbA2 solution and a solution containing HbF. Hereinafter, the solution containing HbF before mixing is also referred to as "HbF solution", and the resulting solution containing HbA2 and HbF is also referred to as "HbA2 / HbF solution". In this embodiment, for example, a reference substance containing HbA2 and HbF with high accuracy can be prepared. Note that the resulting HbA2 / HbF solution may contain components other than HbA2 and HbF (for example, other Hbs such as HbA0 and HbA1, buffers, stabilizers, etc.).

[0034] The origin of the HbF solution is not particularly limited, and examples include blood of humans and non-human animals, and human blood is preferred. Examples of non-human animals are as described above. It is preferable to use an HbF solution obtained by extracting Hb from the blood of the above-mentioned human or non-human animal and performing a purification treatment as necessary. From the viewpoint of containing a large amount of HbF, cord blood is preferred as the blood.

[0035] For example, the HbF solution may be prepared by lysing the blood of the above-mentioned human or non-human animal to extract HbF. Hemolysis may be performed, for example, by adding a buffer solution to the precipitate obtained by centrifuging whole blood obtained by blood collection, and freezing and thawing. Further, the solution obtained by lysing as described above may be centrifuged to recover the supernatant as the HbF solution. Furthermore, the supernatant recovered as described above may be purified by dialysis or the like to obtain the HbF solution.

[0036] The HbF solution preferably contains a buffer solution for adjusting the pH. Examples of the buffer solution include Tris buffer solution (such as Tris-HCl), carbonate buffer solution, HEPES buffer solution, phosphate buffer solution, and the like.

[0037] The HbF solution may contain a stabilizer. Examples of the stabilizer include sugars such as sucrose and trehalose.

[0038] The quantitative ratio of HbA2 to HbF in the HbA2 / HbF solution can be appropriately selected according to the purpose. For example, the quantitative ratio of HbA2 to HbF (HbA2:HbF) may be 1:10 to 10:1, or may be 1:5 to 5:1.

[0039] <Method for manufacturing Hb accuracy control substance> The method for manufacturing an Hb accuracy control substance according to an embodiment of the present disclosure includes bringing a solution having a pH of 7.8 to 9.5 and containing Hb into contact with an anion exchanger. The accuracy control substance is a sample with a known composition used for accuracy control of analysis. In the accuracy control substance of this aspect, it refers to a sample in which the composition of HbA2 and, if necessary, HbF (that is, the amount contained in the accuracy control substance) is known. Details of the steps of the method for manufacturing an Hb accuracy control substance in this embodiment can all be applied to the matters described in the section of "Method for manufacturing a solution containing HbA2". However, replace the terms "solution containing HbA2" or "HbA2 solution" obtained with "Hb accuracy control substance".

[0040] In one aspect, the method for manufacturing an Hb accuracy control substance may further include recovering the solution after contacting it with the anion exchanger. The mode of recovering the solution is as described above in the section of "Method for manufacturing a solution containing HbA2".

[0041] The pH of the Hb solution when contacted with the anion exchanger is 7.8 to 9.5. From the viewpoint of suppressing the binding of HbA2 to the anion exchanger and improving the recovery amount of HbA2, the pH of the Hb solution is preferably 9.0 or less. From the viewpoint of increasing the proportion of HbA2 in the obtained quality control material, the pH of the Hb solution is preferably 8.0 or more, more preferably 8.3 or more, even more preferably 8.5 or more, and particularly preferably 8.8 or more. From the above viewpoints, the pH of the Hb solution is preferably 8.0 to 9.0, more preferably 8.3 to 9.0, even more preferably 8.5 to 9.0, and particularly preferably 8.8 to 9.0.

[0042] In one embodiment, the ratio of HbA2 to the total amount of Hb is increased in the Hb solution after contact with the anion exchanger, compared to the Hb solution before contact with the anion exchanger.

[0043] In one embodiment, the Hb solution comprises Hb derived from the blood of a human subject without Hb abnormalities and a buffer solution. Details of this embodiment are as described above in the section "Method of producing a solution comprising HbA2."

[0044] In one embodiment, the method for producing a quality control material for Hb further comprises mixing the solution obtained by contacting with an anion exchanger with a solution containing HbF. Details of this embodiment are as described above in the section "Production method of a solution containing HbA2 and HbF". However, the obtained term "solution containing HbA2 and HbF" or "HbA2 / HbF solution" is replaced with "quality control material for Hb". EXAMPLES

[0045] Next, the embodiments of the present disclosure will be specifically described using examples, but the embodiments of the present disclosure are not limited to these examples.

[0046] <Example 1: Preparation of quality control material> 1. Preparation of High Concentration HbA2 Solution Peripheral blood collected from normal humans was used as human whole blood. [Hemolysis] (1) Human whole blood was centrifuged (4°C, 10,000 x g, 10 minutes) and the supernatant was discarded. (2) The precipitate was suspended in an amount of saline equal to that of human whole blood. (3) The suspension from step (2) was centrifuged (4°C, 10,000 x g, 10 minutes) and the supernatant was discarded. (4) Steps (2) and (3) were repeated. (5) The precipitate was suspended in 20 mM EDTA-NaOH (pH 7.0) in an amount 1.3 times the volume of the whole blood, and then frozen and thawed. This was used as a crude Hb solution.

[0047] [Purification of crude Hb solution] (1) Ammonium sulfate was added to the crude Hb solution so that the final concentration was 20% by mass. After confirming that it was completely dissolved, the solution was stirred at room temperature for 10 minutes. (2) Centrifugation (4°C, 10,000 x g, 10 minutes) was performed, and the supernatant was collected. (3) The collected supernatant was enclosed in a dialysis membrane with a molecular weight cutoff of 3500, and dialyzed using purified water as the external solution. The external solution was at least 10 times the volume of the internal solution, and the external solution was replaced five times at intervals of at least 3 hours. (4) The internal solution was centrifuged (4°C, 18,600 x g, 10 minutes) and the supernatant was collected. This was designated as solution A.

[0048] [Ion exchange of solution A] (1) A strong anion exchange resin (Macro-PrepHighQ: Biorad) was equilibrated with 20 mM Tris-HCl (pH 8.8). (2) Tris-HCl (pH 8.8) was added to solution A to a final concentration of 20 mM. This is called solution B. (3) Solution B was mixed with an equal amount of anion exchange resin, and the solution was recovered. (4) After the solution was recovered in step (3), an equal amount of 20 mM Tris-HCl (pH 8.8) was added and mixed to the anion exchange resin, and the solution was recovered. (5) After the solution was recovered in step (4), 20 mM Tris-HCl (pH 8.8) containing 150 mM NaCl in an amount equal to that of the anion exchange resin was added, and the solution was recovered. (6) The solutions collected in steps (3), (4), and (5) were mixed in an arbitrary ratio to adjust the HbA2 content. This was designated solution C.

[0049] [Concentration of solution C] (1) Ammonium sulfate was added to solution C, and after confirming that it was completely dissolved, it was stirred at room temperature for 10 minutes. The amount added was 5.18 g per 10 mL of solution C. (2) Centrifuge (4°C, 10,000 x g, 20 minutes) and discard the supernatant. (3) The precipitate was suspended in 37.5 mM Tris-HCl (pH 8.8). This was designated solution D.

[0050] [Dialysis of solution D] (1) Solution D was enclosed in a dialysis membrane with a molecular weight cutoff of 3500, and dialyzed against 37.5 mM Tris-HCl (pH 8.8) as the external solution. The external solution was 30 times the volume of the internal solution, and the external solution was replaced twice at intervals of 24 hours or more. (2) The dialysate was centrifuged (4°C, 40,000 x g, 10 minutes) and the supernatant was collected.

[0051] 2. Preparation of low concentration HbA2 solution (1) A crude Hb solution was obtained by carrying out hemolysis treatment in the same manner as in "1. Preparation of high-concentration HbA2 solution." (2) Ammonium sulfate was added to the crude Hb solution, and after confirming that it was completely dissolved, the solution was stirred at room temperature for 10 minutes. (3) After centrifugation, the supernatant was collected. (4) The recovered supernatant was enclosed in a dialysis membrane with a molecular weight cutoff of 3,500, and dialyzed against purified water as the external solution. (5) The internal solution was centrifuged, and the supernatant was collected. This was designated as solution A. (6) Tris-HCl (pH 8.8) was added to solution A to a final concentration of 37.5 mM.

[0052] 3. Preparation of HbF Solution (1) Human umbilical cord whole blood was subjected to hemolysis treatment in the same manner as in "1. Preparation of high-concentration HbA2 solution" to obtain a crude Hb solution. (2) Ammonium sulfate was added to the crude Hb solution, and after confirming that it was completely dissolved, the solution was stirred at room temperature for 10 minutes. (3) After centrifugation, the supernatant was collected. (4) The recovered supernatant was enclosed in a dialysis membrane with a molecular weight cutoff of 3,500, and dialyzed against purified water as the external solution. (5) The internal solution was centrifuged, and the supernatant was collected. This was designated as solution A. (6) Tris-HCl (pH 8.8) was added to solution A to a final concentration of 37.5 mM.

[0053] 4. Preparation of Quality Control Materials (1) Low-concentration HbA2 solution and HbF solution, and high-concentration HbA2 solution and HbF solution were mixed to prepare two concentrations of HbA2 / HbF solutions. (2) 50% (w / v) sucrose solution was added to each HbA2 / HbF solution at a final concentration of 8.65% (w / v). (3) The obtained quality control material was freeze-dried. The quality control material prepared from the low-concentration HbA2 solution and HbF solution is designated as Lv1, and the quality control material prepared from the high-concentration HbA2 solution and HbF solution is designated as Lv2.

[0054] 5. Evaluation of quality control materials Three lots (Lots 1 to 3) of quality control materials of each concentration were prepared. Using a high performance liquid chromatography device ADAMS HA-8180T (Arkray, Inc.), the percentages (%) of HbA2 and HbF relative to total Hb in the prepared quality control materials were measured by the method described above. Table 1 shows the percentages (%) of HbA2 and HbF relative to total Hb in the raw materials and the prepared quality control materials.

[0055] [Table 1]

[0056] 6. Discussion Three lots of quality control material were prepared using normal adult blood, and HbA2 concentrations were increased in all lots. This method makes it possible to stably prepare HbA2 quality control material from normal blood without using blood from β-thalassemia patients, which is difficult to obtain.

[0057] <Example 2: Study of pH during ion exchange> 1. Experimental Procedure [Preparation] (1) 20 mM Tris-HCl buffer solutions of pH 8.5, pH 8.8, and pH 9.0 were prepared. (2) An ion exchange resin (Macro-PrepHighQ: Biorad) was equilibrated with each buffer solution.

[0058] [Preparation of Hb solution] Peripheral blood collected from normal humans was used as human whole blood. (1) Human whole blood was centrifuged (4°C, 10,000 x g, 10 minutes) and the supernatant was discarded. (2) The precipitate was suspended in an amount of saline equal to that of human whole blood. (3) The suspension obtained in step (2) was centrifuged (4°C, 10,000 x g, 10 minutes), and the supernatant was discarded. (4) Steps (2) and (3) were repeated again. (5) The precipitate was suspended in 20 mM EDTA (pH 7.0) in an amount 1.3 times the volume of human whole blood, and then frozen and thawed. (6) After centrifugation (4°C, 40,000 x g, 10 minutes), the supernatant was collected and used as a crude Hb solution. (7) The Hb solution was enclosed in a dialysis membrane with a molecular weight cutoff of 3,500, and dialyzed using purified water as the external solution. (8) 0.5 M Tris-HCl was added to the dialyzed Hb solution to a final concentration of 20 mM. (9) 20 mM Tris-HCl was added and diluted to a concentration of about 1 / 4 of that of human whole blood. These were used as Hb samples. Three types of Hb samples with different pH values ​​were prepared.

[0059] [Ion exchange] (1) Equal volumes of equilibrated Macro-prep HighQ and Hb sample were mixed. (2) The solution was collected and designated as Supernatant 1. (3) The remaining resin was suspended in 20 mM Tris-HCl in the same volume as the Hb sample in (1). (4) The solution was collected and designated as Supernatant 2. (5) The percentage of HbA2 relative to total Hb in supernatants 1 and 2 was measured.

[0060] 2.Results The ratio (%) of HbA2 to total Hb in supernatants 1 and 2 is shown in Table 2.

[0061] [Table 2]

[0062] 3. Discussion In this study, it was revealed that the percentage of HbA2 in the unbound fractions (supernatants 1 and 2) of the ion exchange increased in a pH-dependent manner. Since the isoelectric point of Hb varies depending on the type, and the binding ability to the ion exchange resin changes depending on the pH, it is considered that under certain conditions, Hb other than HbA2 preferentially binds to the ion exchange resin, resulting in a relatively high percentage of HbA2 in the unbound fraction.

Claims

1. A method for producing a solution containing hemoglobin A2, comprising contacting a solution containing hemoglobin and having a pH of 7.8 to 9.5 with an anion exchanger.

2. The method for producing a solution comprising hemoglobin A2 according to claim 1, further comprising recovering the solution after contact with the anion exchanger.

3. The method for producing a solution containing hemoglobin A2 according to claim 1, wherein the pH of the solution containing hemoglobin is 8.0 to 9.

0.

4. The method for producing a solution containing hemoglobin A2 according to claim 1, wherein the ratio of hemoglobin A2 to the total amount of hemoglobin is increased in the solution after contact with the anion exchanger compared to the solution before contact with the anion exchanger.

5. The method for producing a solution containing hemoglobin A2 according to claim 1, wherein the hemoglobin-containing solution having a pH of 7.8 to 9.5 contains hemoglobin derived from the blood of a human subject without hemoglobin abnormalities and a buffer solution.

6. A method for producing a solution containing hemoglobin A2 and hemoglobin F, comprising mixing a solution containing hemoglobin A2 obtained by the production method according to any one of claims 1 to 5 with a solution containing hemoglobin F.

7. A method for producing a quality control substance for hemoglobin, comprising contacting a solution containing hemoglobin and having a pH of 7.8 to 9.5 with an anion exchanger.

8. The method for producing a hemoglobin quality control material according to claim 7, further comprising recovering the solution after contact with the anion exchanger.

9. The method for producing a hemoglobin quality control material according to claim 7, wherein the pH of the solution containing hemoglobin is 8.0 to 9.

0.

10. The method for producing a hemoglobin quality control material described in claim 7, wherein the ratio of hemoglobin A2 to the total amount of hemoglobin is increased in the solution after contact with the anion exchanger compared to the solution before contact with the anion exchanger.

11. The method for producing a hemoglobin quality control substance according to claim 7, wherein the hemoglobin-containing solution having a pH of 7.8 to 9.5 contains hemoglobin derived from the blood of a human subject without hemoglobin abnormalities and a buffer solution.

12. The method for producing a hemoglobin quality control material according to any one of claims 7 to 11, further comprising mixing the solution obtained by contacting with the anion exchanger with a solution containing hemoglobin F.

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